CD84 Human

What is CD84 and which protein family does it belong to?

CD84 (also known as SLAMF5) is a cell surface receptor belonging to the CD2 subset of the immunoglobulin (Ig) superfamily, specifically within the signaling lymphocyte activation molecule (SLAM) family of receptors. The SLAM family members are characterized by their role in modulating immune responses . Human CD84 is encoded by a gene that produces a 328 amino acid residue precursor protein containing a 21 amino acid signal peptide and a 199 amino acid extracellular domain . Structurally, CD84 contains immunoglobulin-like domains in its extracellular region that are critical for its function in cell-cell interactions and immune signaling pathways .

What is the expression pattern of CD84 in human immune cells?

CD84 demonstrates a distinct expression pattern across immune cell populations, with varying intensity depending on cell type and activation state. It is preferentially expressed on B lymphocytes, monocytes, and platelets . Research also shows that CD84 is expressed on thymocytes and mature T cells, with higher expression on CD45RO+ memory T cells . In the thymic development pathway, CD84 shows highest expression on CD4-CD8- thymocytes, with decreasing expression as cells mature . Additionally, CD84 expression is upregulated on myeloid-derived suppressor cells (MDSCs) and bone marrow stromal cells in certain disease contexts like multiple myeloma . The differential expression patterns suggest cell type-specific regulatory mechanisms and functional roles for CD84.

How does CD84 expression differ between normal and malignant cells?

More dramatically, cells in the tumor microenvironment show substantially enhanced CD84 expression. Bone marrow stromal cells derived from MM patients express higher levels of CD84 compared to those from patients with smoldering disease or healthy donors . Similarly, CD14+ myeloid cells from MM patients show approximately 10-fold higher CD84 expression compared to CD14- cells or CD14+ cells from healthy donors . Both myeloid-derived suppressor cell populations (M-MDSCs and G-MDSCs) from MM patients display significantly elevated CD84 expression compared to cells from earlier disease stages or healthy bone marrow . These expression differences may contribute to the immunosuppressive microenvironment in MM.

What is the ligand for CD84 and how does this interaction occur?

CD84 functions as a self-ligand, engaging in homophilic interactions where CD84 molecules on different cells bind to each other. This homotypic binding characteristic was established through experiments with soluble immunoglobulin fusion proteins containing the human CD84 extracellular domains (CD84-Ig). These fusion proteins were found to bind specifically to CD84-transfected cells, but not to cells expressing other CD2 subfamily receptors .

Mechanistically, the homophilic binding occurs exclusively through the first N-terminal immunoglobulin variable (Ig V) domain of the protein . This was conclusively demonstrated through blocking experiments with anti-CD84 monoclonal antibodies recognizing epitopes within domain 1, which prevented CD84-Ig binding to CD84-expressing cells and platelets . Additional evidence came from studies with CD84 domain human/mouse chimeras, which further confirmed that only the first extracellular domain is involved in ligand-receptor recognition . Importantly, this CD84-CD84 interaction was found to be independent of the molecule's cytoplasmic tail, suggesting that while the extracellular domain mediates binding, the intracellular portion likely handles downstream signaling events .

How does CD84 signaling influence T cell function?

CD84 serves as a co-stimulatory molecule in T cells, enhancing specific immune functions when engaged alongside T cell receptor (TCR) stimulation. Research has demonstrated that concurrent ligation of CD84 (using either monoclonal antibodies or CD84-Ig fusion proteins) and CD3 significantly enhances interferon-gamma (IFN-γ) secretion in human lymphocytes . This suggests CD84 positively modulates T cell effector functions upon activation.

In specific T cell contexts, CD84 has been shown to enhance anti-CD3 induced IFN-γ production in lymphocytes and increase anti-CD3 induced proliferation in phytohemagglutinin (PHA) T cell blasts . These findings indicate CD84 provides additional activation signals that amplify T cell responses. The signaling pathways downstream of CD84 in T cells involve the cytoplasmic adapter protein SAP (SLAM-associated protein), which is encoded by the X-linked lymphoproliferative disease gene . This association with SAP connects CD84 signaling to critical immune regulatory pathways.

At the molecular level, CD84 engagement appears to activate pathways involving AKT and S6 phosphorylation, as demonstrated in other cell types . These pathways are known to regulate various aspects of T cell function, including cytokine production, proliferation, and survival. The timing and context of CD84 engagement likely determine its specific effects on T cell responses.

What methodological approaches can be used to study CD84-CD84 interactions?

Several complementary methodological approaches can effectively study CD84-CD84 homophilic interactions:

• Soluble fusion proteins: Creating CD84-Ig fusion proteins containing the CD84 extracellular domains fused to immunoglobulin constant regions allows for binding studies with flow cytometry. This approach has successfully demonstrated specific binding to CD84-expressing cells but not to cells expressing other CD2 family members .

• Domain mapping with blocking antibodies: Using monoclonal antibodies that recognize specific epitopes within different domains of CD84 can block binding interactions. Studies showed that anti-CD84 mAbs recognizing epitopes within domain 1 blocked CD84-Ig binding to CD84-transfected cells and platelets, confirming domain 1's critical role in homophilic interactions .

• Chimeric receptor constructs: Creating domain-swapped chimeras between human and mouse CD84 or between CD84 and other related proteins allows precise identification of binding interfaces. This approach confirmed that only the first extracellular domain of CD84 is involved in ligand-receptor recognition .

• Cell-based assays with transfected cells: Expressing CD84a in cell lines that normally lack this receptor provides clean systems for binding studies. Experiments with CD84-transfected cells verified the homophilic binding specificity .

• Functional assays: Measuring CD84-dependent cellular responses (e.g., cytokine production or proliferation) after specific manipulation of CD84 interactions provides functional validation. Concurrent ligation of CD84 and CD3 resulting in enhanced IFN-γ secretion demonstrated functional consequences of CD84 engagement .

These methodological approaches provide a comprehensive toolkit for investigating CD84-CD84 interactions at molecular, cellular, and functional levels.

What role does CD84 play in the multiple myeloma microenvironment?

CD84 functions as a critical regulator of the immunosuppressive microenvironment in multiple myeloma (MM). While MM cells themselves express relatively low levels of CD84, cells within the tumor microenvironment show dramatically increased expression . This differential expression pattern suggests CD84 mediates key interactions between malignant cells and supportive stromal and immune cells.

Research has revealed several mechanisms through which CD84 influences the MM microenvironment:

• Regulation of PD-L1/PD-1 expression: CD84 activation induces upregulation of PD-L1 on MM cells and myeloid cells in the microenvironment. Conversely, blocking CD84 using specific antibodies or reducing its expression through siRNA leads to downregulation of PD-L1 at both mRNA and protein levels . This establishes a direct link between CD84 signaling and the PD-L1/PD-1 inhibitory pathway.

• Modulation of T cell responses: The CD84-mediated increase in PD-L1 expression on myeloid cells and PD-1 on T cells creates an immunosuppressive environment that inhibits effective T cell-mediated anti-tumor responses. Blocking CD84 was shown to increase T cell-mediated killing of MM cells, demonstrating its functional importance in regulating anti-tumor immunity .

• Signaling pathway activation: CD84 stimulation activates the AKT and S6 phosphorylation pathways, which are known to regulate PD-L1 expression . This provides mechanistic insight into how CD84 controls the immunosuppressive program.

• Cytokine-mediated regulation: MM cells induce CD84 expression on surrounding cells through secretion of cytokines, particularly macrophage migration inhibitory factor (MIF). MIF binds to CD74 receptors on myeloid cells, triggering increased CD84 expression, which subsequently enhances PD-L1 expression .

These findings position CD84 as a central orchestrator of immune suppression in the MM microenvironment, suggesting its potential as a therapeutic target.

How does CD84 expression change during malignant transformation in B cell disorders?

CD84 expression patterns undergo significant alterations during malignant transformation in B cell disorders, reflecting changes in cell phenotype and function. In the progression from monoclonal gammopathy of unknown significance (MGUS) to multiple myeloma (MM), CD84 expression increases on the malignant plasma cells . This suggests CD84 upregulation may coincide with or contribute to disease progression.

In B cell populations, CD84 is differentially expressed, with CD84hi B cells representing a subset of memory B cells . This normal heterogeneity in expression becomes dysregulated in malignant contexts. In chronic lymphocytic leukemia (CLL), CD84 has been identified as regulating a novel survival pathway, potentially contributing to the characteristic accumulation of malignant B cells .

Furthermore, CD84 mediates interactions between CLL cells and various stromal cells in the microenvironment both in vitro and in vivo . These CD84-mediated cell-cell interactions elevate PD-L1 expression on CLL and surrounding microenvironmental cells, as well as PD-1 expression on T cells, creating an immunosuppressive microenvironment .

The progressive changes in CD84 expression during malignant transformation suggest it could serve as a biomarker for disease progression and a potential therapeutic target in B cell malignancies.

What experimental approaches can be used to investigate CD84's role in tumor immunosuppression?

Several sophisticated experimental approaches can be employed to investigate CD84's role in tumor immunosuppression:

• CD84 blocking antibodies: Utilizing specific blocking antibodies like the B4 monoclonal antibody allows for functional inhibition of CD84-CD84 interactions. This approach has demonstrated that blocking CD84 reduces PD-L1 expression on myeloid cells and enhances T cell-mediated killing of multiple myeloma cells .

• Gene silencing techniques: Employing siRNA targeting CD84 in tumor or immune cells provides a direct approach to assess CD84's functional contributions. Studies have shown that reduced CD84 expression using siRNA results in downregulation of PD-L1 mRNA levels compared to control siRNA-transfected cells .

• CRISPR/Cas9 gene editing: Establishing stable CD84 knockout cell lines using CRISPR/Cas9 technology provides a clean system to study CD84-dependent processes. This approach was successfully used to create CD84-deficient THP1 monocytic cells, which showed downregulation of PD-L1 expression .

• Co-culture systems: Utilizing co-cultures of tumor cells, T cells, and myeloid cells with or without CD84 manipulation allows for assessment of complex cellular interactions. Experiments showing that MM cells induce PD-L1 on CD14+ cells and PD-1 on T cells, effects that were abrogated by anti-CD84 blocking antibodies, highlight the utility of this approach .

• Cytokine manipulation: Determining how specific cytokines (e.g., MIF) modulate CD84 expression provides insight into the regulation of CD84 in the tumor microenvironment. Studies demonstrating that recombinant MIF elevates CD84 and PD-L1 expression on myeloid cells illustrate this approach .

• Signaling pathway analysis: Investigating downstream signaling events following CD84 activation helps elucidate the molecular mechanisms linking CD84 to immunosuppression. Research showing increased phosphorylation of AKT and S6 following CD84 stimulation exemplifies this approach .

• Functional immune assays: Employing cytotoxicity assays (e.g., chromium release assays) and T cell activation assays with CD84 manipulation provides direct assessment of functional outcomes. Studies demonstrating increased 7AAD staining of MM cells and enhanced chromium release when CD84 was blocked illustrate the value of functional readouts .

These complementary experimental approaches provide a comprehensive toolkit for investigating CD84's multifaceted roles in tumor immunosuppression.

How is CD84 expression regulated during T cell development and differentiation?

CD84 expression undergoes dynamic regulation during T cell development and differentiation, suggesting stage-specific functions. In the thymus, CD84 shows highest expression on the most immature CD4-CD8- thymocytes, with expression progressively decreasing as cells mature through the thymic development pathway . This pattern implies potential roles in early T cell developmental processes or selection events.

In the peripheral T cell compartment, CD84 is preferentially expressed on CD45RO+ T cells compared to CD45RA+ populations . Since CD45RO is a marker associated with memory T cells, this suggests CD84 may have particular importance in memory T cell function or maintenance. The differential expression between naive and memory cells indicates CD84 expression may be upregulated following T cell activation and memory formation.

What methodological approaches can be used to study CD84 in lymphoid progenitor identification?

Several methodological approaches are particularly valuable for studying CD84 in the context of lymphoid progenitor identification:

• Highly multiplexed single-cell proteomic screening: This approach allows for comprehensive analysis of cell surface markers, including CD84, alongside other progenitor markers. Recent research applied this technique to human bone marrow progenitors, identifying patterns of CD84 expression within early lymphoid commitment stages .

• Flow cytometry with extended marker panels: Traditional and spectral flow cytometry using panels that include CD84 alongside established hematopoietic stem and progenitor cell (HSPC) markers enables precise characterization of CD84 expression patterns across developmental hierarchies. This approach allows for prospective isolation of specific progenitor populations based on CD84 expression .

• Functional assays with sorted populations: Isolating progenitor populations based on CD84 expression allows for subsequent functional testing through in vitro differentiation assays, colony-forming assays, or in vivo transplantation studies. These approaches can determine whether CD84 expression correlates with or influences lymphoid commitment potential .

• Single-cell transcriptomics: Coupling surface protein expression data with single-cell RNA sequencing provides insights into the transcriptional programs associated with CD84 expression in progenitor populations. This approach can reveal gene regulatory networks potentially influenced by or influencing CD84 expression.

• Lineage tracing: Using genetic approaches to track the fate of CD84-expressing progenitors can determine their developmental potential and contribution to different lymphoid lineages.

• Comparative studies between species: Examining similarities and differences in CD84 expression patterns between human and mouse lymphoid progenitors can identify conserved features and potential species-specific functions .

These complementary approaches provide a comprehensive toolkit for investigating CD84's role in lymphoid progenitor biology and development.

How might targeting CD84 influence immunotherapy outcomes in multiple myeloma?

Targeting CD84 represents a promising strategy that could significantly enhance immunotherapy outcomes in multiple myeloma (MM) through several mechanisms:

• Reversing T cell exhaustion: CD84 signaling induces PD-L1 expression on MM cells and myeloid cells in the tumor microenvironment, contributing to T cell exhaustion. Blocking CD84 reduces PD-L1/PD-1 axis activation, potentially reinvigorating tumor-specific T cell responses . This indicates CD84 blockade could complement or enhance existing checkpoint inhibitor therapies.

• Reprogramming the myeloid compartment: CD84 expression is substantially elevated on immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSCs), in MM patients. Targeting CD84 could reprogram these cells toward a more anti-tumor phenotype by reducing their PD-L1 expression and potentially altering their cytokine profile .

• Disrupting tumor-stroma interactions: CD84 mediates interactions between MM cells and stromal cells in the bone marrow microenvironment. Blocking these interactions could destabilize the supportive niche for MM cells, making them more vulnerable to therapeutic targeting .

• Enhancing T cell-mediated killing: Experimental evidence shows that blocking CD84 elevates T cell-mediated killing of MM cell lines, demonstrated by increased cell death markers and enhanced chromium release in killing assays . This suggests CD84 blockade could potentiate adoptive T cell therapies or CAR-T approaches.

• Targeting cytokine networks: MM cells induce CD84 expression on microenvironmental cells through secretion of MIF, which binds to CD74. This suggests combination approaches targeting both MIF/CD74 and CD84 could more completely disrupt the immunosuppressive network .

These multiple mechanisms position CD84 as a promising therapeutic target that could enhance various immunotherapy approaches in MM through comprehensive reprogramming of the tumor microenvironment.

What technical considerations are important when developing CD84-targeting therapeutic antibodies?

Developing effective CD84-targeting therapeutic antibodies requires careful attention to several technical considerations:

• Epitope selection: The first N-terminal Ig V domain of CD84 is critical for homophilic interactions . Therapeutic antibodies should target specific epitopes within this domain to effectively block CD84-CD84 interactions. Existing blocking antibodies like the B4 mAb provide proof-of-concept for this approach .

• Antibody format optimization: Different antibody formats (full IgG, Fab fragments, single-chain variable fragments) may have distinct advantages for targeting CD84. Full IgG antibodies provide longer half-life and potential effector functions, while smaller formats might offer better tissue penetration, particularly in the bone marrow microenvironment.

• Fc engineering considerations: The Fc portion of anti-CD84 antibodies could be engineered to enhance or suppress specific effector functions (ADCC, CDC, ADCP) depending on the therapeutic goal. Since CD84 is expressed on normal immune cells, minimizing depletion of these populations might be desirable.

• Combination therapy strategies: Since CD84 regulates PD-L1 expression, combining anti-CD84 antibodies with PD-1/PD-L1 inhibitors could produce synergistic effects . Pre-clinical validation of such combinations is essential.

• Biomarker development: Patient selection biomarkers based on CD84 expression patterns or downstream signaling activation could predict response to anti-CD84 therapies. Flow cytometric analysis of CD84 expression on tumor and microenvironmental cells could guide patient selection.

• Cross-reactivity considerations: Since homotypic CD84-CD84 interactions occur between different cell types, therapeutic antibodies must effectively block these interactions in complex cellular environments. This requires antibodies with appropriate affinity and stability in the tumor microenvironment.

• Safety profiling: Given CD84's expression on normal immune cells, careful assessment of on-target, off-tumor effects is essential. In particular, potential impacts on normal B cell, T cell, and myeloid cell functions should be thoroughly evaluated.

These technical considerations highlight the complexities involved in developing CD84-targeting antibodies and underscore the importance of comprehensive preclinical characterization.

How can CD84 expression patterns be leveraged for improved lymphoid malignancy diagnosis?

CD84 expression patterns offer several opportunities for enhancing lymphoid malignancy diagnosis and classification:

• Differential diagnosis of plasma cell disorders: CD84 expression increases during progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) . Quantitative assessment of CD84 expression on plasma cells could potentially serve as an additional marker to distinguish between these conditions or identify MGUS cases at higher risk of progression.

• Characterization of tumor microenvironment: The dramatic upregulation of CD84 on myeloid-derived suppressor cells and bone marrow stromal cells in MM provides an opportunity to assess the immunosuppressive state of the tumor microenvironment . Flow cytometric evaluation of CD84 on these populations could offer insights into disease biology and potentially correlate with treatment response.

• Multi-parameter flow cytometric analysis: Including CD84 in extended antibody panels for flow cytometry could enhance the detection and characterization of various B-cell malignancies. In chronic lymphocytic leukemia (CLL), where CD84 regulates survival pathways, its expression patterns might correlate with disease progression or treatment resistance .

• Identification of memory B-cell derived malignancies: Since CD84hi B cells represent a subset of memory B cells , high CD84 expression could help identify malignancies derived from memory B-cell populations versus those originating from other B-cell developmental stages.

• Assessment of PD-L1/PD-1 axis activation: Given CD84's role in regulating PD-L1 expression, CD84 assessment could serve as a surrogate marker for potential activation of the PD-L1/PD-1 inhibitory axis . This could inform decisions regarding checkpoint inhibitor therapy.

• Minimal residual disease monitoring: If CD84 expression is confirmed as stable on malignant cells, it could potentially be incorporated into flow cytometric panels for minimal residual disease detection after therapy.

By incorporating CD84 assessment into diagnostic algorithms, clinicians could gain additional insights into disease biology, potentially improving diagnostic accuracy, prognostication, and treatment selection for lymphoid malignancies.